Sampling method for accurately evaluating nutrient state of tea tree and application

Abstract

The invention discloses a sampling method for accurately evaluating the nutrient state of a tea tree and application, and the sampling time and leaf positions of mature leaves and new shoots are limited for the first time when the nutrient state of the tea tree is evaluated. The method comprises the following steps: selecting nutrient branches with consistent growth parts on the canopy surface of the tea tree in the previous year, topping the branches at the end of January, and removing top standing buds; in mid-to-late February, the tea tree is still in a dormant period, and a first mature leaf at the top end of a pruning opening is selected. When the dormant bud germinates and develops into one bud and two leaves, taking a new shoot sample corresponding to the marked mature leaves, and not taking the mature leaves at the moment. Through verification of two long-term nitrogen fertilizer positioning tea gardens in 10 years and 20 years and 2000 group samples, the annual relevance of the nutrition state of the tea trees can be ensured by using the method disclosed by the invention, and meanwhile, the fertilization level of the tea gardens can be positively correlated regularly and remarkably in the nutrition state of the tea trees; the young shoot quality component amino acid is positively correlated with the fertilization level remarkably, and the phenol-ammonia ratio is negatively correlated with the fertilization level remarkably.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tea tree cultivation and breeding phenotype identification, and relates to a sampling method for accurately evaluating the nutrient state of tea trees and application thereof. BACKGROUND

[0002] Tea trees are perennial woody plants, and the mature leaf nutrient element contents at different leaf positions on the vegetative branches are quite different. During the spring tea picking period, the element contents of the new shoots at the top of the tea tree canopy are different from those of the new shoots below the canopy, and the nutrient element contents of the new shoots at different development stages, such as the one-bud two-leaf stage and the one-bud three / four-leaf stage, also differ. During field sampling, how to ensure that the samples taken can best represent the nutrient state of tea trees, reflect the fertilization level of the tea garden, and have good repeatability between years without being affected by the climate and environment. SUMMARY

[0003] To solve the above problems, the application designs a sampling method for accurately evaluating the nutrient state of tea trees and application thereof. Through a long-term positioning test of nitrogen fertilizer in a tea garden and verification of nearly 2000 samples of 221 single plants and parent and offspring of the F1 hybrid population of tea trees from 2023 to 2025 for three consecutive years, the method can accurately evaluate the nutrient state of tea trees and better reflect the nitrogen fertilizer application level of the tea garden, has good repeatability between years and is not affected by environmental factors such as climate, and has important significance for accurate identification of the nutrient state of tea trees and the phenotype of tea tree nutrient-efficient varieties.

[0004] The application provides a sampling method for accurately evaluating the nutrient state of tea trees, which comprises three stages of pretreatment before sampling, mature leaf sampling and new shoot sampling.

[0005] Pretreatment before sampling: from January 20 to 28, the overwintering vegetative branches on the shed surface are topped, the top buds are removed, and the positions are marked.

[0006] Mature leaf sampling: from February 20 to 28, the first mature leaf L1 at the top of the marked branch is taken.

[0007] New shoot sampling: after the marked dormant buds germinate and develop to the one-bud two-leaf stage, the new shoot S1 corresponding to the marked mature leaf is taken, and the mature leaf is not taken at this time.

[0008] Measurement index: the nitrogen concentration of the first mature leaf L1 and the new shoot S1 is measured as NL1 and NS1, respectively, in units of mg / g, dry weight; the amino acid A1 of the new shoot S1 sample is measured in units of mg / g, dry weight, and the total amount of tea polyphenols is measured, and the phenolic amino acid ratio P1 is calculated. The mature leaf L1 at the dormant stage and the new shoot S1 at the one-bud two-leaf stage in the long-term positioning site of nitrogen fertilizer are sampled to measure the above indexes, a single linear regression analysis in statistics is adopted, the regression coefficient is determined by the least square method, and finally the nutrient state formula index is obtained, wherein N is the fertilization level: NL1= 0.03433 x N + 24.79; NS1= 0.005839 x N + 51.98; P1= -0.004544 x N + 12.85; A1= 0.009215 x N + 27.01; Combined with the results of long-term positioning experiments in many places for many years, the fertilization level of tea garden is low when the nitrogen fertilizer use amount is 0-200 kg / ha, at this time, the NL1 value is 24.79-31.656, the NS1 value is 51.98-53.1478, the P1 value is 11.9414-12.85, and the A1 value is 27.01-28.853, therefore, when 25<NL1<32, 52<NS1<53, 12<P1<13, and 27<A1<29, it is determined that the fertilization level of tea garden is low, and the tea tree is in a state of poor nutrients; Combined with the results of long-term positioning experiments in many places for many years, the fertilization level of tea garden is high when the nitrogen fertilizer use amount exceeds 600 kg / ha, at this time, the NL1 value is 45.388, the NS1 value is 55.4834, the P1 value is 10.1236, and the A1 value is 32.539, therefore, when NL1>45, NS1>55, P1<10, and A1>32, it is determined that the fertilization level of tea garden is high, and the tea tree is in a state of nutrient surplus.

[0009] In the process of tea garden management, the tea tree shape is maintained through regular pruning every year, and the tea yield and quality of the next year are ensured. The nutrient branches at the top of the canopy are all nutrient branches of the previous year, which can represent the nutrient status of the tea tree in the current year. Previous studies have found that most of the nutrients required for new shoot germination are derived from the corresponding mature leaves. Therefore, the first mature leaf at the top of the canopy of the tea tree best represents the nutrient status of the tea tree. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The total nitrogen content of early spring new shoots, the total amount of amino acids, the phenol-ammonia ratio, and the correlation with the nitrogen application level of tea garden are shown; A: the nitrogen content in early spring new shoots is significantly positively correlated with the nitrogen fertilizer application level of tea garden; B: the content of free amino acids in new shoots is significantly positively correlated with the nitrogen fertilizer application level of tea garden; C: the phenol-ammonia ratio in new shoots is significantly negatively correlated with the nitrogen fertilizer application level of tea garden.

[0011] Figure 2 The total nitrogen content of mature leaves at the end of February, the total nitrogen content of new shoots at the one-bud-two-leaf stage, the nitrogen content, and the nitrogen reutilization efficiency are shown, and the correlation with the nitrogen application level of tea garden is shown; A: the nitrogen content in mature leaves L1 at the end of February during dormancy is significantly positively correlated with the nitrogen fertilizer application level of tea garden; B: the nitrogen content in new shoots at the one-bud-two-leaf stage is significantly positively correlated with the nitrogen fertilizer application level of tea garden; C: the nitrogen content in each new shoot is significantly positively correlated with the nitrogen fertilizer application level of tea garden. 15 15 ​N isotope content was significantly positively correlated with nitrogen fertilizer application level in tea garden; D: nitrogen reutilization efficiency was significantly positively correlated with nitrogen fertilizer application level in tea garden.

[0012] Figure 3 The correlation of mature leaf nitrogen content and fertilizer application level after the new shoots germinated and developed to one bud and two leaves is shown; Figure 4 The annual significant correlation of mature leaf biomass and total nitrogen content in dormant period of 2023, 2024 and 2025 samples is shown; A: the dry weight of each mature leaf of the population per plant in the dormant period was significantly positively correlated between 2023 and 2024; B: the L1 nitrogen content of the mature leaf of the population per plant in the dormant period was significantly positively correlated between 2023 and 2024; C: the dry weight of each mature leaf of the population per plant in the dormant period was significantly positively correlated between 2023 and 2025; D: the L1 nitrogen content of the mature leaf of the population per plant in the dormant period was significantly positively correlated between 2023 and 2025; E: the dry weight of each mature leaf of the population per plant in the dormant period was significantly positively correlated between 2024 and 2025; F: the L1 nitrogen content of the mature leaf of the population per plant in the dormant period was significantly positively correlated between 2024 and 2025.

[0013] Figure 5 The annual significant correlation of mature leaf and all new shoots in the crown layer is shown; A: the L1 nitrogen content of each mature leaf of the population per plant in the dormant period was significantly positively correlated between 2023 and 2024; B: the L1 nitrogen content of the mixed sample of all new shoots in the crown layer of the population per plant was not significantly correlated between 2023 and 2024. T2 DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0015] Please refer to Figures 1-5 The present application provides a sampling method for accurately evaluating the nutrient status of tea plants.

[0016] Select the nutrient branches of the previous year's growth on the tea plant canopy, and prune the branches at the end of January to remove the terminal buds.

[0017] In mid-to-late February, when the tea plants are still in the dormant period, select the first mature leaf at the top of the pruned branches. When the dormant buds germinate and develop to one bud and two leaves, take the sample of the new shoots corresponding to the marked mature leaf, but not the mature leaf at this time. Example 1​

[0018] Samples were taken at the long-term fixed-site nitrogen fertilizer experiment plot (20 years) according to the method described in the present invention to verify whether the method in the present invention can accurately reflect the fertilization level of the tea garden.

[0019] Experimental materials and methods: The long-term fixed-site nitrogen fertilizer plot is located at the Tea Research Institute of the Chinese Academy of Agricultural Sciences (30°10′48.83″N, 120°05′20.49″E). The tea tree variety is "Longjing 43". Since 2005, six nitrogen application levels have been set, and the annual nitrogen fertilizer application amounts are 0, 119, 285, 474, 569, and 712 kg / ha in sequence. Only the new shoots of one bud and two leaves in early spring are collected, the total nitrogen content of the new shoots, the total amino acids and tea polyphenols in the new shoots are analyzed, and the phenol-ammonia ratio is calculated.

[0020] Result analysis: Single linear regression analysis in statistics was adopted, the regression coefficient was determined by the least squares method, and finally the new shoot formula index for determining the nutrient status was obtained, where N is the fertilization level: NS1 = 0.005839×N + 51.98; P1 = -0.004544×N + 12.85; A1 = 0.009215×N + 27.01; According to the experimental results of the long-term fixed-site plot, when the nitrogen fertilizer application amount in the tea garden is 0 - 200 kg / ha, the fertilization level is low. At this time, the NS1 value is 51.98 - 53.1478, the P1 value is 11.9414 - 12.85, and the A1 value is 27.01 - 28.853. Therefore, when 52 < NS1 < 53, 12 < P1 < 13, and 2 < A1 < 29, it is determined that the fertilization level of the tea garden is low and the tea trees are in a poor nutrient state; According to the experimental results of the long-term fixed-site plot, when the nitrogen fertilizer application amount in the tea garden exceeds 600 kg / ha, the fertilization level of the tea garden is high. At this time, the NS1 value is 55.4834, the P1 value is 10.1236, and the A1 value is 32.539. Therefore, when NS1 > 55, P1 < 10, and A1 > 32, it is determined that the fertilization level of the tea garden is high and the tea trees are rich in nutrients.

[0021] According to the method of the present invention, in early spring, the new shoots corresponding to the first mature leaf at the top of the tree crown are taken. The total nitrogen content of the new shoots is significantly positively correlated with the nitrogen application level in the tea garden ( Figure 1 A in Figure 1 ), the total amino acid content of the new shoots is significantly positively correlated with the nitrogen application level in the tea garden ( Figure 1 B in Example 2 <

[0022] A long-term positioning experiment site (10 years) for coupling tea tree varieties and nitrogen fertilizers was used to further verify whether the method of the present invention can indicate the nutritional status of tea trees and reflect the fertilization level of tea plantations.

[0023] Materials and methods: The long-term positioning experiment site for coupling tea tree varieties and nitrogen fertilizers is located at the Shengzhou Base of the Tea Research Institute, Chinese Academy of Agricultural Sciences (120°82′53″ E, 29° 74′83″ N). The tea tree varieties are "Longjing 43" and "Zhongcha 108". Since 2015, three nitrogen application levels have been set, with annual nitrogen application rates of 150, 300, and 450 kg / ha respectively.

[0024] At the end of January, the apical dormant buds were cut off with pruning shears, and some leaves were 15 subjected to N tracer application. In the middle and late February, the first mature leaf under the pruning cut was collected. The one-bud-two-leaf shoot was the one-bud-two-leaf shoot germinated from the dormant bud corresponding to the first mature leaf under the pruning cut. Analyze the total nitrogen content and 15 N content of the mature leaves at the dormant stage (L1) and the one-bud-two-leaf shoots, and calculate the nitrogen recycling efficiency, which is the 15 N content in the shoots divided by the total 15 N amount applied.

[0025] Result analysis: Single linear regression analysis in statistics was used to determine the regression coefficient by the least squares method, and finally the formula index for determining the nutrient status of mature leaves at the dormant stage was obtained, where N is the fertilization level: NL1 = 0.03433×N + 24.79; Combining the experimental results of two long-term positioning sites in Example 1 and Example 2, when the nitrogen fertilizer application rate in the tea plantation is 0 - 200 kg / ha, the fertilization level is low. At this time, the NL1 value is 24.79 - 31.656. Therefore, when 25 < NL1 < 32, it is determined that the fertilization level of the tea plantation is low and the tea trees are in a nutrient-poor state; when the nitrogen fertilizer application rate in the tea plantation exceeds 600 kg / ha, the fertilization level of the tea plantation is high. At this time, the NL1 value is 45.388. Therefore, when NL1 > 45, it is determined that the fertilization level of the tea plantation is high and the tea trees are nutritionally sufficient.

[0026] The total nitrogen content of the mature leaves collected at the end of February, that is, the mature leaves at the dormant stage, is extremely significantly positively correlated with the fertilization level of the tea plantation ( Figure 2 A in), and the total nitrogen content of the one-bud-two-leaf shoots is extremely significantly positively correlated with the fertilization level of the tea plantation ( Figure 2 B in). After 15 N labeling of the mature leaves of tea trees at the dormant stage in late January, it can indicate the redistribution of nitrogen in the mature leaves to the shoots. The 15 N content in the shoots is significantly positively correlated with the nitrogen application level of the tea plantation ( Figure 2 C in), and the corresponding nitrogen recycling efficiency is significantly positively correlated with the nitrogen application level of the tea plantation ( Figure 2(D in the text). Additionally, analysis of the correlation between nitrogen content and fertilization level in mature leaves at the one bud and two leaves stage after new shoot emergence revealed that the nitrogen nutrient status of mature leaves at this stage was unrelated to fertilization level. Figure 3 The above results demonstrate that the sampling method of the present invention can accurately reflect the nutrient status of tea trees and its correlation with the fertilization level of tea gardens. Example 3

[0027] The sampling method of this invention was used to sample 223 individual plants and parent plants of the F1 generation hybrid population of tea trees for three consecutive years to verify that the sampling method of this invention can ensure strong correlation between the years of sampling and is not affected by environmental and climatic factors.

[0028] According to the method of the present invention, the first mature leaf at the top of the canopy of 223 individual plants was collected in mid-to-late February of 2023, 2024 and 2025, and the total nitrogen content and biomass were measured. Figure 4 It can be seen that the dry weight of each mature leaf during the dormant period of a single plant in the population was significantly positively correlated between 2023 and 2024. Figure 4 A) shows a significant positive correlation between the two years 2023 and 2025. Figure 4 C) shows a significant positive correlation between 2024 and 2025. Figure 4 The nitrogen content in L1 of mature leaves during dormancy was significantly positively correlated between 2023 and 2024. Figure 4 (B in the equation) shows a significant positive correlation between 2023 and 2025. Figure 4 (D in the original text); significantly positively correlated between the two years 2024 and 2025 ( ). Figure 4 (F in the text). Therefore, the biomass and total nitrogen of the 2023, 2024, and 2025 samples were significantly correlated across years, and the nutrient status of the samples showed good repeatability. Simultaneously, samples of all new shoots in the tea canopy and mature leaves after shoot emergence were collected. The nutrient status of samples collected during this period could not be reproduced across years. The L1 level of each mature leaf at the one-bud-two-leaf stage on a single plant in the population was [not specified]. T2 There was no correlation between dry weight in 2023 and 2024. Figure 5 In section A), the nitrogen content of all new shoots in the canopy mixed sample showed no correlation between the two years of 2023 and 2024. Figure 5 (B) indicates that there is no correlation between the nutrient content of samples across years. Figure 5 Therefore, only the dormant mature leaves described in this invention can be used for precise identification of population nutrient utilization phenotypic traits.

[0029] Therefore, the method in this invention can accurately evaluate the nutrient status of tea trees, providing a fast, efficient and accurate method for the field of tea tree cultivation and breeding phenotypic identification.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is limited by the appended claims and their equivalents.

Claims

1. A sampling method for accurately evaluating the nutrient status of tea trees, characterized in that: It includes three stages: pre-sampling treatment, sampling of mature leaves, and sampling of new shoots; Pre-sampling treatment: On January 20 - 28, top the overwintering vegetative branches on the shed surface, remove the apical dormant buds, and mark the positions; Sampling of mature leaves: On February 20 - 28, take the first mature leaf L1 at the top of the marked branches; Sampling of new shoots: After the marked dormant buds germinate and develop to one bud with two leaves, take the new shoot S1 corresponding to the marked mature leaf. At this time, do not take the mature leaf.

2. The sampling method for accurately evaluating the nutrient status of tea trees according to claim 1, characterized in that, Determination indexes: Measure the nitrogen concentrations of the first mature leaf L1 and new shoot S1 samples as NL1 and NS1, with the unit of mg / g, dry weight; the amino acid A1 of the new shoot S1 sample, with the unit of mg / g, dry weight, and the total amount of tea polyphenols, and calculate the phenol-ammonia ratio P1; Collect mature leaf L1 and new shoot S1 samples from the long-term fixed-position plot of nitrogen fertilizer. Use single linear regression analysis in statistics to determine the regression coefficient by the least squares method, and finally obtain the formula index for judging the nutrient status, where N is the fertilization level: NL1 = 0.03433×N + 24.79; NS1 = 0.005839×N + 51.98; P1 = -0.004544×N + 12.85; A1 = 0.009215×N + 27.01; When 25 < NL1 < 32, 52 < NS1 < 53, 12 < P1 < 13, 27 < A1 < 29, it is determined that the fertilization level of the tea garden is low and the tea plants are in a nutrient-deficient state; When NL1 > 45, NS1 > 55, P1 < 10, A1 > 32, it is determined that the fertilization level of the tea garden is high and the tea plants are nutrient-rich.

3. Application of the sampling method for accurately evaluating the nutrient status of tea plants according to claim 1 or 2, characterized in that: The mature leaves in the dormant period are applied to the accurate identification of the phenotypic traits of population nutrient utilization.

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